Synthetic conjugate of CpG DNA and T-help/CTL peptide

ABSTRACT

Highly effective vaccine compositions are constructed according to the methods of this invention. The methods are amenable to use with any peptidic antigen sequence and involve covalent attachment of an immunostimulatory nucleotide sequence to an antigenic peptide sequence. Preferred antigenic peptides are fusion peptides made up of one or more CTL epitope peptides in sequence fused to a T helper peptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/963,173 filed on 26 Apr. 2018, which is a continuation of U.S. patentapplication Ser. No. 14/736,897 filed on 11 Jun. 2015, now U.S. Pat. No.9,974,854, which is a division of U.S. patent application Ser. No.11/008,958 filed on 13 Dec. 2004, now U.S. Pat. No. 9,090,673, which inturn is related to and claims benefit of U.S. provisional patentapplication Ser. No. 60/528,706 filed on 12 Dec. 2003. Each applicationis hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under grant numbersAI044313 and CA030206, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention. .

SUBMISSION OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled1954578US3SequenceListing.txt, created on 5 Feb. 2020 and is 5 kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION Technical Field

This invention relates to the field of immunology. In particular, theinvention involves methods for presenting immunogenic substances to thebody in a manner that results in enhanced recognition of the substanceby the immune system. The compounds and methods of the inventionconsequently are useful for preparation of vaccines, methods ofvaccinating and vaccination for both protection from and treatment fordiseases such as bacterial and viral diseases or any disease amenable toprevention or treatment by vaccine, including cancer. Diseasespreviously resistant to attempts to vaccinate against them, such as HIVdisease are of particular interest in uses for the invention.

Description of the Background Art

Long-term administration of anti-viral drugs is expensive and ofteninvolves unpleasant or even dangerous side effects. Increasing numbersof pathogenic bacteria are becoming drug-resistant. Treatment ofinfectious disease in the current health care environment thereforeoften is problematic. Moreover, prevention of disease, where possible,is far preferable to treatment. Therefore, non-toxic and effectivevaccines against a variety of pathogens would be highly desirable.However, no vaccine, approved as safe and effective by the U.S. Food andDrug Administration, currently exists for a number of diseases,including HIV. Even where vaccines for a particular pathologic agent doexist, more potent, more effective and safer vaccines are needed in theart.

A vaccine that can stimulate CTL and a T-help cellular immune responsecan be used both as a prophylactic vaccine and also as part of atreatment for those who are infected. Stimulation of CTL is important inrestricting viral replication during both the acute and chronic stagesof infection; these CTL responses are critical in the immunologicaldefense against such diseases as HIV, including resistance to infectionin the first instance and long-term non-progression to AIDS in infectedpersons. T helper responses also are necessary for an optimal immuneresponse to any infectious disease. A vaccine which effectively producesan increase in both CTL and T helper immune responses would be ofenormous use for treatment or prophylaxis of any number of diseases,particularly viral diseases such as HCMV and HIV.

Exogenous T helper activity can be provided in trans byco-administration of the pan HLA DR-binding epitope, PADRE. The PADREsequence is a chemically defined promiscuous T helper peptide epitopecapable of binding with high affinity to a broad range of the mostcommon HLA-DR types. Vaccines using this strategy are known to requireformulation with a potent adjuvant to evoke a cytolytic response.Previously, peptide administered with DNA adjuvant has been shown tostimulate CTL in a T_(h)-independent manner, but in some cases onlyafter repeated doses.

To produce a strong CTL response, it is usually necessary to formulate avaccine with powerful immunological adjuvants. Unfortunately, many ofthe known adjuvants fail to induce antigen-specific CTL, and many haveassociated side effects which make them unsuitable for human use.Recently, DNA adjuvants have attracted attention as safe and effectivefor human use with the ability to promote CTL responses. Adjuvantactivity has been associated with palindromic DNA sequences that containunmethylated CpG dinucleotides which conform to the general consensusmotif of XCGY, where X is any base except C and Y is any base except G.Increasing the number of stimulatory CpG motifs in anoligodeoxynucleotide (ODN) increases its activity, while the addition ofa CpG on an end or in an unfavorable sequence context could actuallyreduce the degree of immune activation. Krieg et al., Nature374:6546-6549, 1995. Elimination of the CpG dinucleotides from ODNabolish their stimulatory activity. When CpG is replaced with GpG, theODN becomes inhibitory and antagonistic to the activity of the parentODN. Hoe et al., J. Immunol. 171:4920-4926, 2003.

For activating human cells, the optimal motif is GTCGTT and the best CpGmotif for mouse or rabbit immune cells is two 5′ purines followed by theCpG dinucleotide and ending with two 3′ pyrimidines. The CpG DNAdirectly stimulates antigen presenting cells to produce cytokines(including TNF-α, IL-1, IL-6, IL-10, IL-12 and GM-CSF) and to upregulateexpression of MHC and crucial costimulatory molecules. CpG DNA also mayact on B lymphocytes, inducing their proliferation and then productionof IL-6 and IL-10. CpG ODN also caused enhanced cytotoxicity andenhanced IFN-γ secretion by NK cells. The effect on NK cells may beindirect, requiring the presence of adherent cells of CpG-conditionedsupernatants which contain IL-12, TNF-α and Type 1 interferons.

Interspecies differences in recognition by toll-like receptors (TLR) orother immune activation recognition may result in differences inrecognition of CpG motifs. For example, the mouse TLR9 molecule ispreferentially activated by the CpG motif GACGTT, whereas the human TLR9is optimally triggered by the motif GTCGTT. See Bauer et al., Proc.Natl. Acad. Sci. USA 98:9237-9242, 2001; Hartmann et al. J. Immunol.164:1617-1624, 2000. Despite these differences, addition of a CpG ODN toa commercial hepatitis B vaccine markedly accelerated seroconversion(protective IgG antibody levels attained in 2 weeks), showing theusefulness of these types of strategies in humans. See Krieg, TrendsImmunol. 23:64-65, 2002. CpG adjuvant activity (increased rapidity ofresponse and higher titers, resulting in protective titers to hepatitisB in 89% of the subjects by 8 weeks) has also been shown inimmunocompromised HIV-infected patients. This beneficial effect in apopulation with reduced response can be extended to benefit other groupssuch as the elderly, alcoholics and cancer patients, as well as thegeneral population. Therefore, to add extra insurance that positiveimmune responses seen in the well-known mouse model systems arepredictive of the same results in humans, experimentation with ahuman-specific or a primate- and human-specific CpG ODN sequence shouldconfirm the techniques usefulness.

Without wishing to be bound by theory, TLR9 ligands (CpG ODN) may exerttheir effects by specifically and strongly stimulating plasmacytoiddendritic cells, which are thought to have an important role in T-cellself-tolerance to antigens. See Krieg, Nat. Med. 9(7):831-835, 2003.Adjuvants that can activate plasmacytoid dendritic cells through theTLR9 receptor may be one key to get the most out of a vaccine antigen.See Kuwana et al., Eur. J. Immunol. 31:2547-2557, 2001; Ferguson et al.,J. Immunol. 168:5589-5595, 2002.

Current strategies have not yielded vaccines that induce a strong,durable CTL response which results in protection from or prevention ofmany diseases, including, for example HIV, HCMV and cancer, which couldbe useful in public health, either to prevent occurrence of disease orto promote immune attack in affected persons. Therefore, new methods ofproducing vaccines are needed in the art.

SUMMARY OF THE INVENTION

Accordingly, embodiments of the invention provide a conjugated vaccinemolecule which comprises an antigenic peptide and a DNA oligomer. TheDNA oligomer may comprise a CpG sequence, such as a phosphodiester (PO)CpG DNA or a fully phosphorothioated (PS) backbone CpG DNA orcombinations thereof which include PO and PS linkages. DNA oligomerssuitable for use in the invention may comprise about 8 to about 300nucleotide bases, preferably about 15 to about 100 nucleotide bases, andmost preferably about 20 to about 25 nucleotide bases. The antigenicpeptide preferably comprises a CTL epitope, such as PADRE:I9V (SEQ IDNO:1), KSS:PADRE:S9L, (SEQ ID NO:2) or PADRE:I9V:K/N:S9L (SEQ ID NOs:3and 4)(see Table XXI), and may comprise a fusion peptide comprisingPADRE and a CTL peptide epitope sequence. Preferably, the antigenicpeptide is about 8 to about 50 amino acid residues or more, including75, 100 or 150 amino acid residues, or any number that can besynthesized practically using automated methods.

Yet further embodiments of the invention provide an improvement in apeptide vaccine composition which comprises conjugating the peptide to aDNA oligomer and a method of increasing the effectiveness of a peptidevaccine component which comprises conjugating the peptide to a DNAoligomer.

Preferably, the peptide portion comprises minimal cytotoxic epitopes andT-help epitopes that have been defined as immunogenic by variousimmunologic analyses and that provide an optimal fit into an HLA Class Ior Class II molecule's peptide binding groove, depending on their type.The combination of refined peptides and DNA still function as a vaccine,and the components do not inactivate each other, which would not havebeen predicted a priori from first principles.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical synthetic scheme for the synthesis ofConjugates 1 and 2.

FIG. 2 is a polyacrylamide gel. Lane 1=Oligo 1; lane 2=Oligo 2; lane3=Conjugate 1; lane 4=Conjugate 2; lane 5=the maleimide propionicaddition product of AKXVAAWTLKAAAILKEPVHGV (X=cyclohexylalanine; SEQ IDNO:1).

FIG. 3 shows the chemical synthetic scheme for the synthesis ofConjugates 3-7.

FIG. 4 is a polyacrylamide gel. Lane 1=Oligo 3; lane 2=Conjugate 2; lane3=Conjugate 3; lane 4=Hyd-PADRE:I9V (AKXVAAWTLKAAAILKEPVHGV;X=cyclohexylalanine; SEQ ID NO:1).

FIG. 5 shows percent lysis by splenocytes immunized once with 1 nmoleConjugate 3 or 50 nmole PADRE:I9V (AKXVAAWTLKAAAILKEPVHGV;X=cyclohexylalanine; SEQ ID NO:1) P (T≤t) two-tail 0.03.

FIG. 6 shows a partial synthetic scheme for synthesis of Conjugate 4(SEQ ID NOs:2 and 9).

FIG. 7 is a polyacrylamide gel. Lane 1=Oligo 3; lane 2=Conjugate 2.

FIG. 8 shows a partial synthetic scheme for synthesis of Conjugate 5(SEQ ID NOs:4 and 9).

FIG. 9 is a polyacrylamide gel. Lane 1=Oligo 3; lane 2=Conjugate 6; lane3=Conjugate 5.

FIG. 10 shows a partial synthetic scheme for synthesis of Conjugate 6(SEQ ID NOs:3 and 9).

FIG. 11 is a polyacrylamide gel. Lane 1=Oligo 3; lane 2=Conjugate 6;lane 3=Conjugate 5.

FIG. 12 shows percent lysis of human HIV-infected CD4⁺ T cells(diamonds, JA2; triangles and squares, R7). P (T≤t) two-tail 0.03.

FIG. 13 shows response to challenge by surrogate virus after a singleimmunization with 5 nmoles Conjugate 3 versus CpG alone. Each barprovides data from a single mouse. Each treatment was performed on threemice.

FIG. 14 shows response to challenge by recombinant vaccinia virusexpressing the HIV-pol gene after a single intranasal administration of1 or 10 nmole Conjugate 3 versus 10 nmole CpG DNA or PADRE:I9V peptidealone. (P≤0.02, Wilcoxon Two Sample Test). Each bar represents data froma single mouse. Each treatment was performed on two or three mice asshown.

FIG. 15 shows response to challenge by recombinant vaccinia virusexpressing HIV-pol after subcutaneous and intraperitoneal immunizationwith 1 nmole Conjugate 3 or 1 nmole PADRE:I9V peptide plus CpG DNA. Eachbar provides data from a single mouse. Each treatment was performed onthree mice.

FIG. 16 shows response to challenge by recombinant vaccinia virusexpressing HIV-pol after immunization with 0.1 nmole Conjugate 3 orpeptide+DNA, fusion peptide or CpG DNA alone. Each bar provides datafrom a single mouse. Each treatment was performed on multiple mice asshown.

FIG. 17A-C is a set of three FACS results of cells stained to identifyIFN-γ after immunization with Conjugate 4 (17C), peptide mix (17B) orDNA alone (17A).

FIG. 18 shows percent cell lysis of targets by immune splenocytes afterimmunization with 0.1 nmole Conjugate 5 (Squares) or its individualcomponents (diamonds). Targets were pre-loaded with I9V peptide(ILKEPVHGV; SEQ ID NO:5). P<0.03.

FIG. 19 shows percent cell lysis of targets by immunized splenocytesafter immunization with 0.1 nmole Conjugate 5 (Circles) or itsindividual components (Squares). Targets were pre-loaded with S9Lpeptide (SLYNTVATL; SEQ ID NO:6). P<0.01.

FIG. 20 shows cell lysis of R7 cells that were infected with HIV bysplenocytes immunized with Conjugate 5 (Bars) or with its individualcomponents (Squares). P<0.01.

FIG. 21A-D provides histograms of the flow cytometry analysis of immunesplenocytes for reaction with an isotype control (21A), an irrelevant,control peptide (21B), Conjugate 5 (21C) or fusion peptide plus DNA,unconjugated (21D).

FIG. 22 shows results of a chromium release assay performed usingI9V-loaded JA2 cells as targets and cells from HHD II mice immunizedwith either Conjugate 7 or Conjugate 3.

FIG. 23 presents percent cytotoxicity data for cells from HLA A2/Kb miceimmunized with either Conjugate 3 or Conjugate 10.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention provides an efficient means to immunize or vaccinateagainst infectious disease or any other disease treatable or preventableby vaccination, for example, HIV, HCMV and cancer. The inventioninvolves a method of covalently attaching an antigenic peptide (forexample a human cytomegalovirus, HIV or cancer antigen) to a DNAsequence that acts as an adjuvant, enhancing response to the antigen.Such DNA sequences, known as immunostimulatory nucleotide sequences, areper se known and discussed in U.S. Pat. No. 6,514,948, the disclosuresof which are hereby incorporated by reference.

This invention involves covalent attachment of an immunostimulatorynucleic acid, such as a cytosine-phosphate-guanosine (CpG)-containingDNA sequence, to an antigenic peptidic sequence. The conjugates of thisinvention may be synthesized by reacting a DNA having a 5′-end thiohexylmodification with an N-terminal maleimide propionic acid-modifiedpeptidic sequence to form a conjugate or by reacting DNA having a5′-aldehyde modification to a peptide with a N-terminal hydrazine toform a conjugate linked by a hydrazone linkage. Preferred methods fortheir construction are illustrated in Examples 1-5. The fused vaccinecompositions of this invention enhance the immune response in a mammalto the antigenic peptidic sequence and thereby increase theeffectiveness of the antigen as a vaccine.

Such DNA-peptide fusions may include any DNA sequence that acts toincrease immune stimulation. DNA oligomers of any source are suitable,including bacterial, viral, insect, mammalian or any source at all,including synthetic DNA molecules and DNA molecules with eitherphosphodiester or phosphorothioate backbones. The particular sequencecontained in the DNA is not of paramount importance because any sequencecan be effective to stimulate immunity according to this invention,however nucleotide sequences with CpG DNA are preferred.

In general, single-stranded DNA oligomers are used, preferably about 20to about 25 nucleotides long, however, double-stranded DNA iscontemplated for use with this invention. Double-stranded DNA conjugatescan be made by first attaching single-stranded DNA to the peptide andthen hybridizing the resulting conjugate to a complementarysingle-stranded DNA sequence. Longer or shorter DNA sequences may beused with this invention, including DNA sequences from about 8 to about300 nucleotides long, or from about 15 to about 100 nucleotides long.Any convenient size or sequence of DNA may be used with this invention,however, it is generally preferred to use single-stranded DNA of about10, 15, 20, 25, 30, 35, 40, or 50 nucleotides in length. DNA containingCpG sequences is preferred, but not necessary. Exemplary nucleic acidsinclude, but are not limited to 5′-tcgtcgllllgtcgllllgtcgtt-3′(phosphorothioate-substituted; SEQ ID NO:10) and5′-ggGGGACGATCGTCgggggG-3′ (in which phosphorothioate linkages aredenoted by lower case letters and phosphodiester linkages by upper caseletters; SEQ ID NO:11). See Hartmann et al., J. Immunol. 164:1617-1624,2000. Sequences having fully phosphorothioated backbones, partiallyphosphorothioated backbones, or fully phosphodiester backbones aresuitable.

In recent years, CpG DNA have been grouped into different classes calledCpG-A and CpG-B by the Krieg group and CpG-D and CpG-K by Klinman andcolleagues. See Verthelyi et al., J. Immunol. 166(4):2372-2377, 2001;Krug et al., Eur. J. Immunol. 31(7):2154-2163, 2001; Klinman et al.,Microbes. Infect. 4(9):897-901, 2002; Gursel et al., J. Leukoc. Biol.71(5):813-820, 2002. Any of these CpG sequences are suitable for usewith this invention.

An ODN known to have immunologic activity is referred to as ODN 2216.This is a mixed phosphodiester-phosphorothioate backbone of the sequenceggGGGACGATCGTCgggggG (SEQ ID NO:11) with small letters denotingphosphorothioate linkages and uppercase letters denoting phosphodiesterlinkages. This ODN is CpG A-class as defined by Krieg and collaborators.Additional motifs include pyrimidine NTTTTGT and its derivatives asdescribed by Elias et al., J. Immunol. 171:3697-3704, 2003. In thecontext of induction of a T-helper 2 response, Tamura et al. havedescribed ODN without CpG which function to elicit those classes ofresponses. These responses are not necessarily stimulatory for the typesof CTL response here. Sano et al., J. Immunol. 170:2367-2373, 2003.

The peptide moiety of the DNA-peptide compounds according to thisinvention may be any antigenic peptide from any source against whichenhanced immune response is desired. It is contemplated that theinvention will be used most frequently with viral and bacterialantigens, particularly from bacteria and viruses that are causativeagents in infectious disease. Therefore, the invention may be used tocreate vaccine compositions for treatment and prophylaxis of suchdisease as HIV, human cytomegalovirus, tuberculosis or any infectionsdisease. Peptide epitopes of bacterial or viral origin are especiallypreferred. As well, cancer antigens may be used in the present inventionto enhance the efficacy of cancer vaccines, for either treatment orprophylaxis.

The peptides may be of any convenient length so long as the antigenicfunction of the peptide is intact. Peptide CTL epitopes generally areabout 8 to about 12 amino acids long, and therefore such peptides arecontemplated for use with the invention and are preferred. Peptidesshorter than 8 amino acids long, for example 5, 6 or 7 amino acids long,are not preferred but may be used, however longer peptide sequences aremost suitable, including 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50,100 or more amino acids in length, including multi-epitope peptides. SeeAlexander et al., J. Immunol. 168(12):6189-6198, 2002), the disclosuresof which are hereby incorporated by reference.

Fusion peptides including an antigenic peptide as described above fusedto another peptide sequence are specifically contemplated for use withthe inventive methods and compounds. Especially preferred peptidesinclude fusion peptides composed of a CTL epitope sequence and a helperT lymphocyte (CD4) epitope sequence fused together. Examples of suitableCD4 epitopes include the synthetic sequence PADRE, tetanus-specificpeptides, peptides derived from the same antigen or other antigens fromthe virus that is to be targeted. Similarly, for cancer antigens, a CD4peptide derived from the same antigen, or any other cell-antigen knownin the art, and the like may be used. Linker peptide sequences at the N-or C-terminal end of the fusion or between the CTL and CD4 epitopes inthe fusion also may be used. Such linker sequences generally are fromabout 1 to about 10 amino acids in length and preferably are about 2 toabout 7 amino acids in length or more preferably from about 3 to about 5amino acids in length and may comprise modified or non-traditional aminoacids.

Conjugate vaccine compounds may be made by providing amaleimide-substituted or hydrazine-substituted antigenic peptide of theformula

wherein B is an antigenic peptide having about 8 to about 50 or moreamino acid residues; providing a 5′-end thiohexyl modified CpG DNA ofthe formula A-(CH₂)₆—SH or an aldehyde (Amidite-A™) of the formulaA-Amidite-A™, wherein A is a CpG DNA oligomer having about 8 to about300 nucleotide bases; reacting the peptide of step (a) and the DNA ofstep (b); and purifying the resulting conjugate vaccine compound of theformula

wherein B is an antigenic peptide having about 8 to about 50-100 or moreamino acid residues and A is a CpG DNA oligomer having about 8 to about300 nucleotide bases. Compounds made by this method also form part ofthis invention. Preferred compounds and methods are those in which theantigenic peptide is a fusion peptide comprising PADRE and a CTL peptideepitope sequence. The CpG DNA oligomer may have a phosphodiester orfully phosphorothioated backbone.

The biologic properties of peptides were studied in HLA A*0201/Kb mice.These model mice express the human HLA type and are well-known tosuccessfully predict human clinical immune responses and demonstrate theusefulness of these molecules in modifying immunity to the antigen. Thenon-natural assembly of these peptide-DNA fusions was processed toresult in an immunologic response with greater sensitivity ofrecognition compared to non-linked molecules.

All references cited herein are incorporated by reference into thisspecification in their entirety. In light of the preceding description,one skilled in the art can practice the invention in its full scope. Thefollowing examples, therefore, are to be construed as illustrative onlyand not limiting in relation to the remainder of the disclosure.

EXAMPLES Example 1 Synthesis of Peptide-Oligonucleotide (CpG DNA)Conjugates 1 & 2

Synthesis of Conjugate 1 (a conjugate of CpG DNA Oligo 1 (SEQ ID NO:7)and PADRE:I9V fusion peptide (SEQ ID NO:1)) and Conjugate 2 (a conjugateof CpG DNA Oligo 2 (SEQ ID NO:8) and PADRE:I9V fusion peptide (SEQ IDNO:1)) was accomplished as follows. See FIG. 1.

(1) Synthesis of Mal-Peptide

All reagents were Peptide Synthesis Grade. Synthesis: Fmoc-Strategy,Manual. Wang™ Resin (substitution about 0.8 mmole/g) (Novabiochem™, SanDiego, Calif.).

Fmoc-Val-OH was attached to the Wang™ resin (1 eq; substitutionapproximately 0.8 mmole by the symmetrical anhydride (5 eq)/DMAP (1 eq)method. The symmetrical anhydride was made using Fmoc-Val-OH and1,3-diisopropylcarbodiimide (DIC) in a 2:1 ratio. A total of 0.4 gFmoc-Val-Wang™ Resin (approximately 0.22 mmole) was produced. The Fmocgroup was deblocked at each step of the growing peptide chain by 20%piperidine/DMF treatment. All the amino acids used during chainelongation were N^(α)-Fmoc-protected, however, amino acids with sidechain functionalities were used with the additional protective groups:(a) histidine modified with trityl; (b) lysine and tryptophan modifiedwith Boc; (c) glutamic acid and threonine modified with t-butyl.Coupling of all amino acids was done using2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium (HBTU)/DIEAreagents. Peptide chain elongation was continued using known methods tocreate the PADRE-I9V epitope fusion peptide (AKXVAAWTLKAAAILKEPVHGV;X=cyclohexylalanine; SEQ ID NO:1). After deprotection of the Fmoc groupfrom the N-terminal alanine of the peptide chain assembled on the resin,maleimide propionic acid was introduced to the free amino group of thealanine in the presence of DIC/1-hydroxybenzotriazole reagent.

The remaining protecting groups then were removed. Final cleavage wasachieved by treating the peptide-linked resin with trifluoroacetic acid(TFA)/triisopropylsilane (TIS)/water (95/2.5/2.5) for two hours at roomtemperature. This was followed by methyl-t-butyl ether (MBTE)precipitation, filtration and drying of the product.

Approximately 440 mg crude product (about 40% pure Mal-peptide) offormula was obtained (X=cyclohexylalanine; SEQ ID NO:1).

Purification was performed on a Shimadzu™ HPLC System consisting ofLC-8A binary pumps and a SPD-10A UV detector using a VYDAC column(22×250 mm; packing material: C-4, 10μ, 300 Å). About 440 mg crudepeptide (purity about 40%) was purified in 2 lots. Each lot of about210-220 mg was dissolved in 40 ml of 20% acetic acid/H₂O, filteredthrough a 0.45μ, filter, and loaded on a pre-equilibrated column througha solenoid valve (FCV-11AL; Shimadzu™) from port B, attached to the lineof LC-8A Pump-A.

With detection at a wavelength of 220 nm, one column-volume of mobilephase A (about 80 mL) was run through the column at 8 ml/minute Mobilephase A consisted of 0.05% or 0.10% TFA in water filtered through 0.2μfilter paper. A gradient of mobile phase A and mobile phase B (0.08% TFAand in acetonitrile/water (1:1)) then was run as shown in Table I,below, at 8 ml/minute.

TABLE I HPLC Mobile Phase, Step One Gradient Conditions Time (min.) % A% B 0.01 80 20 50 55 45 60 0 100 70 0 100 70.01 80 20 80 Stop

Fractions were collected. The peptide material eluted between 30 and 45minutes. The desired fractions were pooled and subjected to analyticalHPLC. The mobile phases were as described above, with a flow rate of 1ml/minute. The column was a 4.6×250 mm C-18 column (5μ, 300 Å). Afterloading onto a pre-equilibrated column as described above, the gradientshown in Table II, below was run.

TABLE II HPLC Mobile Phase, Step Two Gradient Conditions Time (min.) % A% B 0.01 50 50 25 0 100 30 0 100 30.01 50 50 39 Stop

All fractions containing purified peptide were pooled and concentratedto one-third their original volume using a Rotavapor™, then freeze-driedin a pre-weighed vial. About 70 mg peptide was obtained.

This freeze-dried material was analyzed for purity using the same HPLCmethodology as described above for step two and also by the followingmethod. A sample was loaded onto a 4.6×250 mm C-4 column (5μ, 300 Å) andthe gradient provided in Table III, below was run. Mobile phase C was10% acetonitrile in 0.1 M triethylammonium acetate, pH 5.2-5.4 in water.Mobile phase D was 100% acetonitrile. By both HPLC methods, thesynthetic peptide was greater than or equal to 85% pure.

TABLE III HPLC Mobile Phase, Analysis. Gradient Conditions Time (min.) %C % D 0.01 100 0 20 40 60 25 40 60 25.01 100 0 30 Stop

The synthetic peptide also was analyzed by mass spectroscopy on aShimadzu-Kratos™ MALDI-TOF™ Kompact Probe™ instrument. The determinedmass was within the stated error of the instrument (calculated mass=2478amu, experimental mass=2477.9 amu).

Oligo 1 (phosphodiester single-stranded CpG DNA(5′-tccatgacgttcctgacgtt-3; SEQ ID NO:7), with the 5′ end modified tothiohexyl) and Oligo 2 (fully phosphorothioated backbone,single-stranded CpG DNA (5′-tccatgacgttcctgacgtt-3′ (SEQ ID NO:8) withthe 5′ end modified to thiohexyl, (—(CH₂)₆—S—H) were analyzed byanalytical HPLC according to method B above and found to be about 85%and 60% pure, respectively. The DNAs each were dissolved in 6.5 ml 0.1 Mtriethylammonium acetate, pH 7.0.

Synthesis of Conjugate 1

The conjugate reaction was carried out between PADRE:I9V (SEQ ID NO:1)and Oligo 1 above. All reagents were DNA synthesis grade. The peptide(5.0 mg, 2 eq) was dissolved in 0.1 M guanidine hydrochloride solution(5 mL), pH 6.5-7.0. Oligo 1 (6.5 mg, 1 eq) was dissolved in 0.1 M TEAA(6.5 mL), pH 7.0. The above two solutions were mixed together under N₂atmosphere and stirred for 4 hours at room temperature. The reactionmixture was filtered through a 0.45μ filter and passed through thepreparative HPLC C-4, 10×250 mm, column for desalting and purification(flow rate=4 mL/min; λ=260 nm; mobile phase A=10% ACN, 0.1M, TEAA, pH5.2-5.4; mobile phase B=ACN). Mobile phase A and B were filtered through0.2μ filter paper. The column was equilibrated with one column volume(about 20 mL) mobile phase A. After loading the sample, the column wasdesalted with one column volume (about 20 mL). The desired product waseluted using the following gradient. See Table IV.

TABLE IV HPLC Mobile Phase, Synthetic Conjugate Gradient Conditions Time(min.) % E % D 0.01 100 0 30 40 60 35 40 60 35.01 0 100 45 0 100 45 Stop

The desired peak eluted at between 20-30 minutes of the gradient run.The peak was collected and concentrated on a Rotavapor™ to ⅓ of itsoriginal volume, then freeze dried in a pre-weighed vial. About 3.0 mgof Conjugate 1 was obtained. The product was analyzed by HPLC usingMethod B as above and 8 M urea, 15% acrylamide PAGE as follows. The gelwas run at 250 V for 30 minutes. The sample (10 μL) was prepared informamide, heated to 70_(C)C for 5 minutes and loaded onto the gel withbromophenol blue tracking dye. The gel then was run at 250 V for aboutthirty minutes until the dye reached the bottom. The gel was stainedusing Gel Code Blue™ Stain Reagent. See FIG. 2.

Synthesis of Conjugate 2

The conjugate reaction was carried out between PADRE:I9V peptide (SEQ IDNO:1) and Oligo 2. Peptide (2.5 mg, 1 eq) was dissolved in 2 M urea (5mL). Oligo 2 (6.5 mg, 1 eq) was dissolved in 2 M urea (6.5 mL). Theabove two solutions were mixed together under N₂ atmosphere and stirredfor four hours at room temperature. The reaction mixture was filteredthrough a 0.45μ filter and passed through a preparative HPLC column fordesalting and purification: flow rate=4 mL/minute; λ=260 nm; mobilephase A=10% ACN, 0.1 M TEAA, pH 5.2-5.4; mobile phase B=ACN. Mobilephases A and B were filtered through a 0.2μ (Millipore) filter paper.The column was equilibrated with one column volume (about 20 mL) and thesample was loaded, then desalted with one column volume (about 20 mL).The gradient below was used to elute the desired product at between20-30 minutes.

TABLE V HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.01100 0 30 40 60 35 40 60 35.01 0 100 45 0 100 45 Stop

The desired peak was collected and concentrated on a Rotavapor™ to ⅓ ofits original volume, then freeze-dried in a pre-weighed vial. About 2.0mg of Conjugate 2 was obtained. The product was analyzed by HPLCaccording to Method B (70-80% pure), and by PAGE according to Method Cand produced a single band. See FIG. 2.

Example 2 Synthesis of Peptide-Oligonucleotide (CpG DNA) Conjugate 3

Synthesis and purification of Hyd-PADRE:I9V (SEQ ID NO:1) conjugate witha fully phosphorothioated backbone, single-stranded CpG DNA(5′-tccatgacgttcctgacgtt-3; SEQ ID NO:9) with 5′ end modified toaldehyde, obtained from Trilink Technologies™ and referred to as Oligo3, was accomplished as follows. All the reagents were peptide synthesisgrade. Fmoc-Val-OH (>99%) was attached to the Wang™ resin (substitutionabout 0.8 mmole/g; 1 eq) by the symmetrical anhydride (5 eq)/DMAP (1 eq)method. Synthesis proceeded as using Fmoc-Val-OH and DIC in a 2:1 ratio.See FIG. 3.

Synthesis was started with 0.4 g Fmoc-Val-Wang™ resin (approximately0.22 mmole). The Fmoc group was deblocked at each step of the growingpeptide chain by 20% piperidine/DMF treatment. All the amino acids usedduring chain elongation were N^(α)-Fmoc-protected, however amino acidswith side chain functionalities were used with the additional protectivegroups: (histidine modified with trityl; lysine and tryptophan modifiedwith Boc; glutamic acid and threonine modified with t-butyl). Couplingof all amino acids was done using HBTU/DIEA reagents. After deprotectionof the Fmoc group from the N-terminal alanine of the peptide chainassembled on the resin, HDA was introduced to the free amino group ofalanine in the presence of DIC/1-hydroxybenzotriazole reagent. Finalcleavage was done by treating peptide-resin with TFA/TIS/water in a95/2.5/2.5 ratio for two hours at room temperature. This was followed byMBTE precipitation, filtration and drying of the product. Approximately440 mg of crude product (about 40% purity by analytical HPLC, Method A)was obtained.

Purification was done on Shimadzu™ HPLC system with LC-8A binary pumpsand SPD-10A UV Detector, using a VYDAC column (22×250 mm; packingmaterial, C-4, 10μ, 300 Å). About 440 mg crude peptide (purity about40%) was purified in 2 lots. Each lot of about 210-220 mg was dissolvedin 40 ml of 20% acetic acid/H₂O, filtered through a 0.45μ filter, andloaded on a pre-equilibrated column through the solenoid valve FCV-11ALfrom port B, attached to the line of LC-8A Pump-A (flow rate=8mL/minute; λ=220 nm; mobile phase=0.05% TFA, H₂O; acetonitrile). Mobilephases A and B were filtered through 0.2μ filter paper. The column wasequilibrated with one column volume of mobile phase A (about 80 mL) andthe sample loaded. One column volume of mobile phase A (about 80 mL) wasfollowed by the gradient below.

TABLE VI HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.0180 20 50 55 45 60 0 100 70 0 100 70.01 80 20 80 Stop

The desired peak eluted between 30 and 45 minutes of the gradient. Thefractions were analyzed by analytical HPLC according to Method A: flowrate=1 mL/minute; λ=220 nm, column=C-18, 5μ, 300 Å, 4.6×250 mm; mobilephase A=0.10% TFA, H₂O; mobile phase B=0.08% TFA, 1:1 ACN/H₂O.

TABLE VII HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.0150 50 25 0 100 30 0 100 30.01 50 50 39 Stop

All the fractions containing pure peptide 85%) from all the four lotswere pooled and concentrated on a Rotavapor™ to ⅓ of its originalvolume, then freeze-dried in a pre-weighed vial. About 80 mg of peptidewas obtained. This freeze dried material was again analyzed byanalytical HPLC and mass analysis by method A above and method B: flowrate=1 mL/minute; λ=260 nm; column=C-4, 5μ, 300 Å, 4.6×250 mm; mobilephase A=10% acetonitrile, 0.1 M TEAA, pH 5.2-5.4; mobile phase B=ACN.

TABLE VIII HPLC Mobile Phase Gradient Conditions Time (min.) % A % B0.01 100 0 20 40 60 25 40 60 25.01 100 0 0 Stop

Mass analysis was carried out on a Shimadzu™-Kratos™ MALDI-TOF™ KompactProbe™ mass analyzer. The calculated mass was 2462 a.m.u. Theexperimental mass was 2462 a.m.u., which is within the stated error ofthe instrument.

CpG-DNA fully phosphorothioated with 5′-end modified to aldehyde (Oligo3) was obtained from Trilink Technologies™, analyzed by analytical HPLCas per Method B above and found to be ≥60% pure.

Synthesis of Conjugate 3

The peptide above, PADRE:I9V (SEQ ID NO:1; 4.2 mg, about 1.7 eq), wasdissolved in 2 M urea solution (5 mL). Oligo 3 (SEQ ID NO:8; 13.4 mg,about 2 eq) was dissolved in 2 M urea (15 mL). The above two solutionswere mixed together and stirred for three hours at room temperature. Thereaction mixture was filtered through a 0.45μ filter and passed throughthe preparative HPLC C-4, column (22×250 mm) for desalting andpurification: (flow rate=8 mL/minute; λ—260 nm; mobile phase A=10% ACN,0.1 M TEAA, pH 5.1-5.2; mobile phase B=ACN). Mobile phases A and B werefiltered through a 0.2μ filter paper. The column was equilibrated withone column volume (about 80 mL) of mobile phase A and the sample loaded.One column volume (about 80 mL) was run through the column fordesalting. The following gradient was used to elute the desired product.

TABLE IX HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.01100 0 50 50 50 50.01 0 100 60 Stop

The desired peak eluted between 35-45 minutes of gradient run. The peakcollected above was concentrated on a Rotavapor™ to ⅓ of its originalvolume and then freeze dried in a pre-weighed vial. About 5.1 mg ofConjugate 3 was obtained. The product was analyzed by HPLC by Method Babove and found to be 70-80% pure. Under PAGE, a single band was foundusing Method C: gel, 8 M urea, 15% acrylamide; pre-run at 250 V for 30minutes; sample run, 10 μL sample formamide, heated to 70_(C)C for fiveminutes and loaded; tracking dye, bromophenol blue; run at 250 V untiltracking dye reaches bottom (about 30 minutes). The gel was stained withGel Code Blue™ Stain Reagent. See FIG. 4.

Synthesis of Conjugate 3 (Batch-2)

All reagents were DNA synthesis grade. Hyd-PADRE:I9V (SEQ ID NO:1; 4.1mg, about 1.7 eq) was dissolved in 2 M urea solution (5 mL). Oligo 3(SEQ ID NO:8; 13.5 mg, about 2 eq) was dissolved in 2 M urea (15 mL).The above two solutions were mixed together and stirred for three hoursat room temperature. The reaction mixture was filtered through a 0.45μfilter and passed through the preparative HPLC diphenyl column (10×250mm) for desalting and purification: flow rate=4 mL/minute; λ=260 nm;mobile phase A=10% ACN, 0.1 M TEAA, pH 5.1-5.2; mobile phase B=ACN.Mobile phases A and B were filtered through a 0.2μ filter paper. Thecolumn was equilibrated with one column volume (about 20 mL) of mobilephase A and the sample loaded. One column volume (about 20 mL) was runthrough the column for desalting. The gradient below was used to elutethe desired product.

TABLE X HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.01100 0 40 40 60 45 0 100 60 Stop

The desired peak eluted between 20-30 minutes of the gradient run. Thepeak collected above was concentrated on Rotavapor™ to ⅓ of its originalvolume and then freeze dried in a pre-weighed vial. About 7.0 mg ofConjugate 3 was obtained. The product was analyzed by HPLC by Method Babove, using a diphenyl column, in place of the C-4 column and found tobe 70-80% pure. The material formed a single band by PAGE, Method C.

To demonstrate that the conjugate molecules are immunogenic, HLA A2/kbmice were immunized once subcutaneously with 1 nmole Conjugate 3.Fourteen days later, the spleens were retrieved. The CTL responses ofthe immune splenocytes were tested after one round of in vitrostimulation (IVS). FIG. 5 shows that administration of a single dose of1 nmole Conjugate 3 results in 80% target cell lysis at an E:T ratio of100, while administration of two doses of 50 nmoles PADRE:I9V fusionpeptide (unconjugated; AKXVAAWTLKAAAILKEPVHGV; X=cyclohexylalanine; SEQID NO:1) resulted in only 8% cell lysis at the same E:T ratio. Targetswere labeled with I9V peptide (SEQ ID NO:5) for both sets of animals.This demonstrates the ability of Conjugate 3 to be processed and toinduce specific CTL responses. This experiment was repeated four timeswith similar results each time. Using a two-tailed T test, there was asignificant difference of P<0.03 at all E:T ratios. An additionalanalysis using flow cytometry revealed that high frequencies of splenicT cells were stimulated by the incubation with the I9V peptide toproduce IFN-γ. This IFN-γ-producing population is present only afterstimulation with the specific I9V peptide. Consistently, between 5 and60% of the CD8⁺ T cell population were specific for I9V afterimmunization with Conjugate 3.

Example 3 Synthesis of Peptide-Oligonucleotide (CpG DNA) Conjugate 4

Synthesis and purification of Hyd-KSS:PADRE: S9L; KSSAKXVAAWTLKAAASLYNTVATL; X=cyclohexylalanine; (SEQ ID NO:2; PADRE-HIVgag fusion) wasperformed as follows. Fully phosphorothioated backbone, single-stranded(ss) CpG DNA (5′-CHO—C₆H₅—(CONH—(CH₂)₆—O— tccatgacgttcctgacgtt-3; SEQ IDNO:9) with 5′ end modified to aldehyde was obtained from TrilinkTechnologies™ and termed Oligo 3 in the text. See FIGS. 3 and 6.

Synthesis of Conjugate 4

The Conjugate reaction was carried out between Hyd-KSS:PADRE: S9L and5′-aldehyde substituted oligonucleotide with phosphorothioated backbonei.e. Oligo 3.

Synthesis of Hyd-KSS:PADRE: S9L

All the reagents were Peptide Synthesis Grade. Synthesis: Fmoc-Strategy,Manual. Wang™ resin (substitution about 0.8 mmole/g). Fmoc-Leu-OH (>99%)was attached to the Wang™ resin (1 eq) by the symmetrical anhydride (5eq)/DMAP (1 eq) method as described above (symmetrical anhydride wasmade using Fmoc-Val-OH and DIC in a 2:1 ratio. Synthesis was startedwith 0.4 g Fmoc-Leu-Wang™ resin (approximately 0.22 mmole). TheFmoc-group was deblocked at each step of the growing peptide chain by20% piperidine/DMF treatment. All the amino acids used during chainelongation were N^(α)-Fmoc-protected, however, amino acids with sidechain functionalities were used with additional protective groups:asparagine modified with trityl; lysine and tryptophan modified withBoc; tyrosine, serine and threonine modified with t-butyl. Coupling ofall amino acids was done using HBTU/DIEA reagents. After deprotection ofthe Fmoc-group from the N-terminal lysine of the peptide chain assembledon the resin, HDA (1 eq) was introduced to the free alpha amino group oflysine in the presence of DIC/1-hydroxybenzotriazole reagent. Finalcleavage was done by treating the peptide-resin withTFA/thioanisole/ethanedithiol/water in a 90/5/2.5/2.5 ratio (2 hourtreatment) at room temperature. This was followed by MTBE precipitation,filtration and drying of the product. Approximately 400 mg of crudeproduct (about 40% Purity, analyzed by analytical HPLC, Method A) wasobtained.

Purification of Hyd-Peptide

Purification was done on Shimadzu HPLC system consisting of LC-8A binarypumps and an SPD-10A UV Detector, using VYDAC column (22×250 mm; packingmaterial, C-4, 10μ, 300 Å). About 400 mg crude peptide (purity about40%) was purified in 2 lots. About 200 mg was dissolved in 40 mL 20%acetic acid/H₂O, filtered through a 0.45μ filter, and loaded onto apre-equilibrated column through a solenoid valve FCV-11AL (Shimadzu™)from port B, attached to the line of LC-8A Pump-A: flow rate=8mL/minute; λ=220 nm; mobile phase=0.05% TFA, H₂O; acetonitrile (ACN).Mobile phases A and B were filtered through a 0.2μ filter paper. Thecolumn was equilibrated with one column volume of mobile phase A (about80 mL) and the sample was loaded as above. One column volume of MobilePhase A (about 80 ml) was run through the column, followed by thegradient below.

TABLE XI HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.0180 20 50 55 45 60 0 100 70 0 100 70.01 80 20 80 Stop

The desired peak eluted between 30 and 45 minutes of the gradient run.The fractions were analyzed by analytical HPLC as per Method A: flowrate=1 mL/minute; λ=220 nm; column=C-18, 5μ, 300 Å, 4.6×250 mm; mobilephase A=0.10% TFA, H₂O; mobile phase B=0.08% TFA, 1:1 ACN/H₂O.

TABLE XII HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.0150 50 25 0 100 30 0 100 30.01 50 50 39 Stop

All the fractions containing pure peptide (85%) from all the four lotswere pooled and concentrated on a Rotavapor™ to ⅓ of its originalvolume, then freeze-dried in a pre-weighed vial. About 60 mg of peptidewas obtained. This freeze dried material was analyzed by analytical HPLC(method A above and method B below) and mass analysis. Mass analysis wascarried out on Shimadzu-Kratos™ MALDI-TOF™ Kompact Probe™ mass analyzer.The calculated mass was 2754 a.m.u. and the experimental mass was 2754.4a.m.u., which is within the stated error of the instrument. HPLC methodB: flow rate=1 mL/minute; λ=260 nm; column=diphenyl, 5μ, 300 Å, 4.6×250mm; mobile Phase A=10% ACN, 0.1 M TEAA, pH 5.0-5.1; mobile phase B=ACN.

TABLE XIII HPLC Mobile Phase Gradient Conditions Time (min.) % A % B0.01 100 0 20 40 60 25 40 60 25.01 100 0 0 Stop

Oligo 3

CpG-DNA fully phosphorothioated with 5′-end modified to aldehyde (CHO)was obtained from Trilink Technologies™, analyzed by analytical HPLC bymethod B above and found to be about 60% pure.

Synthesis of Conjugate 4

All the reagents were DNA synthesis grade. Hyd-KSS-PADRE:S9L (SEQ IDNO:2; 4.7 mg, about 1.7 eq) was dissolved in 2 M urea solution (5 mL).Oligo 3 (14.7 mg, about 2.2 eq) was dissolved in 2 M Urea (15 mL). Thetwo solutions were mixed together and stirred for 3 hours at roomtemperature. The reaction mixture was filtered through a 0.45μ filterand passed through a preparative HPLC diphenyl column (10×250 mm) fordesalting and purification: flow rate=4 mL/min; λ=260 nm; mobile phaseA=10% ACN, 0.1 M TEAA, pH 5.0-5.1; mobile phase B=ACN. Mobile phases Aand B were filtered through 0.2μ filter paper. The column wasequilibrated with one column volume (about 20 mL) mobile phase A, andthe sample was loaded. One column volume (about 20 mL) was run throughthe column for desalting. The gradient below was used to elute thedesired product.

TABLE XIV HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.01100 0 40 40 60 45 0 100 60 Stop

The desired peak eluted between 20-30 minutes of the gradient run. Thedesired peak collected above was concentrated on a Rotavapor™ to ⅓ ofits original volume and then freeze dried in a pre-weighed vial. About6.0 mg of Conjugate 4 was obtained. This product was analyzed by HPLC(about 70-80% pure by method B above) and by PAGE (forming a single bandby method C: gel preparation, 8 M urea, 15% acrylamide; pre-run, 250 Vfor 30 minutes; sample run, 10 μL sample formamide, heated to 70_(C)Cfor 5 minute and loaded, tracking dye, bromophenol; run, 250 V for about30 minutes or until the dye reached the bottom; staining, Gel Code Blue™Stain Reagent. See FIG. 7.

Example 4 Synthesis of Peptide-Oligonucleotide (CpG DNA) Conjugate 5

Synthesis and purification of Hyd-PADRE:I9V:N: S9L (AKXVAAWTLKAAAILKEPVHGVNSLYNTVATL; X=cyclohexylalanine; SEQ ID NO:4;PADRE-HIVpol-Asn-HIVgag fusion) was performed as follows. Fullyphosphorothioated backbone, single-stranded CpG DNA (5′-3′tccatgacgttcctgacgtt; SEQ ID NO:9) with 5′ end modified to aldehyde(CHO) was obtained from Trilink Technologies™ and is termed Oligo 3. Theconjugate reaction was carried out between Hyd-PADRE:I9V:N:S9L (SEQ IDNO:4) and Oligo 3. See FIG. 8.

Synthesis of Hyd-PADRE:I9V:N:S9L

All the reagents were Peptide Synthesis Grade. Synthesis: Fmoc-Strategy,manual, using Wang™ resin (substitution about 0.8 mmole/g. Fmoc-Leu-OH(>99%) was attached to the Wang™ resin (1 eq) by the symmetricalanhydride (5 eq)/DMAP (1 eq) method as described above. The symmetricalanhydride was made using Fmoc-Val-OH and DIC in a 2:1 ratio. Thesynthesis was started with 0.4 g Fmoc-Leu-Wang™ resin (approximately0.22 mmole). The Fmoc group was deblocked at each step of the growingpeptide chain by 20% piperidine/DMF treatment. All the amino acids usedduring chain elongation were N^(α)-Fmoc-protected, however, amino acidswith side chain functionalities were used with the following additionalprotective groups: asparagine and histidine modified with trityl; lysineand tryptophan modified with Boc; glutamic acid, tyrosine, serine andthreonine modified with t-butyl. Coupling of all amino acids was doneusing HBTU/DIEA reagents. After deprotection of the Fmoc-group from theN-terminal alanine of the peptide chain assembled on the resin, HDA (1eq) was introduced to the free amino group of alanine in the presence ofDIC/HOBt reagent.

Final cleavage was achieved by treating the peptide-resin withTFA/thioanisole/EDT/water in a 90/5/2.5/2.5 ratio (2 hour treatment) atroom temperature. This was followed by MTBE precipitation, filtrationand drying of the product. Approximately 450 mg of crude product (about30% purity as determined by analytical HPLC, method A) was obtained.Purification was achieved using a Shimadzu HPLC system consisting ofLC-8A binary pumps and a SPD-10A UV detector, using a VYDAC column(2×250 mm; packing material, C-4, 10μ, 300 Å) Sample was prepared asfollows. Four hundred fifty milligrams crude peptide (purity about 40%)was purified in 2 lots. For each lot, 220-230 mg peptide was dissolvedin 40 mL of 20% acetic acid/H₂O and filtered through 0.45μ filter, thenloaded on a pre-equilibrated column through a solenoid valve FCV-11AL(Shimadzu™) from port B, attached to the line of LC-8A Pump-A: flowrate=8 mL/minute; λ=220 nm; mobile phase A, 0.05% TFA, H₂O; mobile phaseB, ACN. Mobile phases A and B were filtered through 0.2μ filter paper.One column volume of Mobile Phase A (about 80 mL) was used toequilibrate the column. The sample was loaded as described above. Onecolumn volume of mobile phase A (about 80 mL) was run through the columnfor desalting, followed by the gradient below.

TABLE XV HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.0187 13 50 62 38 60 0 100 70 0 100 70.01 87 13 80 Stop

The desired peak eluted between 40 and 55 minutes of the gradient run.The collected fractions were analyzed by analytical HPLC per method A:flow rate=1 mL/minute; λ=220 nm; column=C-18, 5μ, 300 Å, 4.6×250 mm;mobile phase A=0.10% TFA, H₂O; mobile phase B=0.08% TFA, 1:1 ACN/H₂O.

TABLE XVI HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.0150 50 25 0 100 30 0 100 30.01 50 50 39 Stop

All fractions containing pure peptide (A5%) from all the four lots werepooled and concentrated on a Rotavapor™ to ⅓ of its original volume,then freeze-dried in a pre-weighed vial. About 60 mg of peptide wasobtained. This freeze dried material was analyzed by analytical HPLC(method A above and method B below) and mass analysis using aShimadzu-Kratos™ MALDI-TOF™ Kompact Probe™ mass analyzer.

The calculated mass was 3539 a.m.u. and the experimental mass was 3539.2a.m.u., which is within the stated error of the instrument. Method B:flow rate=1 mL/minute; λ=260 nm; column=diphenyl, 5μ, 300 Å, 4.6×250 mm;mobile phase A=10% ACN, 0.1 M TEAA, pH 5.0-5.1; mobile phase B=ACN.

TABLE XVII HPLC Mobile Phase Gradient Conditions Time (min.) % A % B0.01 100 0 20 40 60 25 40 60 25.01 100 0 0 Stop

Oligo 3

CpG-DNA fully phosphorothioated with 5′-end modified to aldehyde wasobtained from Trilink Technologies™, analyzed by analytical HPLC byMethod B above and found to be ≥60% pure.

Synthesis of Conjugate 5

All the reagents were DNA synthesis grade. Hyd-PADRE:I9V:N:S9L (SEQ IDNO:4; 7.2 mg, about 2 eq) was dissolved in 2M urea solution (7 mL).Oligo 3 (17.0 mg, about 2.5 eq) was dissolved in 2 M urea (17 mL). Thetwo solutions were mixed together and stirred for 3 hours at roomtemperature. The reaction mixture was filtered through a 0.45μ filterand passed through the preparative HPLC diphenyl column (10×250 mm) fordesalting and purification: flow rate=4 mL/min; λ=260 nm; mobile phaseA=10% ACN, 0.1 M TEAA, pH 5.0-5.1; mobile phase B=ACN. Mobile phases Aand B were filtered through 0.2μ filter paper. The column wasequilibrated with one column volume (about 20 mL) of mobile phase A andthe sample loaded. One column volume (about 20 mL) was run through thecolumn for desalting. The following gradient then was used to elute thedesired product.

TABLE XVIII HPLC Mobile Phase Gradient Conditions Time (min.) % A % B0.01 100 0 40 40 60 45 0 100 60 Stop

The desired peak eluted between 20-32 minutes of the gradient run. Thepeak collected above was concentrated on a Rotavapor™ to ⅓ of itsoriginal volume and then freeze dried in a pre-weighed vial. About 8.0mg of Conjugate 5 was obtained. The product was analyzed by HPLC(≥70-80% pure by method B above) and by PAGE (method C), where it ran asa single band. Method C: 8 M urea, 15% acrylamide gel; pre-run at 250 Vfor 30 minutes; Samples prepared with 10 μL sample formamide, heated to70_(C)C for 5 minutes and loaded with bromphenol blue tracking dye. Thegel was run at 250 V for about 30 minutes or until the dye reached thebottom. Staining was done with Gel Code Blue™ Stain Reagent. See FIG. 9.

Example 5 Synthesis of Peptide-Oligonucleotide (CpG DNA) Conjugate 6

Synthesis and purification of Hyd-PADRE:I9V:K:S9L (AKXVAAWTLKAAAILKEPVHGVKSLYNTVATL; SEQ ID NO:3). The conjugate reaction carried out betweenHyd-PADRE:I9V:K:S9L and Oligo 3 (described above). See FIG. 10.

Synthesis of Hyd-PADRE:I9V:K:S9L

All the reagents were Peptide Synthesis Grade. Synthesis: Fmoc-Strategy,manual. Wang™ resin (substitution about 0.8 mmole/g). Fmoc-Leu-OH (>99%)was attached to the Wang™ resin (1 eq) by the symmetrical anhydride (5eq)/DMAP (1 eq) method as described above using Fmoc-Val-OH and 1,3-DICin a 2:1 ratio. The synthesis was started with 0.4 g Fmoc-Leu-Wang™resin (approximately 0.22 mmole). The Fmoc group was deblocked at eachstep of the growing peptide chain by 20% piperidine/DMF treatment. Allthe amino acids used during chain elongation were Nα-Fmoc-protected,however amino acids with side chain functionalities were used with theadditional protective groups: asparagine and histidine modified withtrityl; lysine and tryptophan modified with Boc; glutamic acid,tyrosine, serine and threonine modified with t-butyl. Coupling of allamino acids was done using HBTU/DIEA reagents. After deprotection of theFmoc group from the N-terminal alanine of the peptide chain assembled onthe resin, HDA (1 eq) was introduced to the free amino group of alaninein the presence of DIC/HOBt reagent.

Final cleavage was achieved by treating the peptide-resin withTFA/thioanisole/EDT/water in a 90/5/2.5/2.5 ratio (2 hour treatment) atroom temperature. This was followed by MTBE precipitation, filtrationand drying of the product. Approximately 450 mg of crude product (about30% purity, by analytical HPLC, method A) was obtained.

Purification of Hyd-Peptide

Purification was done on a Shimadzu™ HPLC system consisting of LC-8Abinary pumps and a SPD-10A UV detector, using a VYDAC column (22×250 mm,packing material, C-4, 10μ, 300 Å). Sample was prepared as follows.Approximately 450 mg crude peptide (purity about 40%) was purified in 2lots. Each lot of about 220-230 mg peptide was dissolved in 40 mL 20%acetic acid/H₂O and filtered through a 0.45μ filter, then loaded onpre-equilibrated column through solenoid valve FCV-11AL (Shimadzu™) fromport B, attached to the line of LC-8A Pump-A: flow rate=8 mL/minute;λ=220 nm; mobile phase=0.05% TFA, H₂O; mobile phase B=ACN. Mobile phasesA and B were filtered through 0.2μ filter paper. The column wasequilibrated with one column volume of mobile phase A (about 80 mL) andthe sample was loaded as described above. One column volume of mobilephase A (about 80 mL) was run through the column, followed by thegradient below.

TABLE XIX HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.0180 20 50 55 45 60 0 100 70 0 100 70.01 87 13 80 Stop

The desired peak eluted between 25 and 40 minutes of the gradient run.The collected fractions were analyzed by analytical HPLC by Method A.All fractions containing pure peptide 85%) from all the four lots werepooled and concentrated on a Rotavapor™ to ⅓ of its original volume,then freeze-dried in a pre-weighed vial. About 70 mg of peptide wasobtained. This freeze-dried material was analyzed by analytical HPLC(methods A 85% pure) and B 85% pure) as described above) and massanalysis using a Shimadzu-Kratos™ MALDI-TOF™ Kompact Probe™ massanalyzer. The calculated mass was 3553 a.m.u. and the experimental masswas 3553.3 a.m.u., which is within the stated error of the instrument.

Oligo 3

CpG-DNA fully phosphorothioated with 5′-end modified to aldehyde wasobtained from Trilink Technologies™, analyzed by analytical HPLC as perMethod B above and found to be about 60% pure.

Synthesis of Conjugate 6

All the reagents were DNA synthesis grade. Hyd-PADRE:I9V:K:S9L (SEQ IDNO:3) (5.8 mg, about 1.6 eq) was dissolved in 2 M urea solution (6 mL).Oligo 3 (13.4 mg, about 2.0 eq) was dissolved in 2 M urea (14 mL). Thetwo solutions were mixed together and stirred for 3 hours at roomtemperature. The reaction mixture was filtered through a 0.45μ filterand passed through the preparative HPLC diphenyl column (10×250 mm) fordesalting and purification: flow rate=4 mL/min; λ=260 nm; mobile phaseA=10% ACN, 0.1 M TEAA, pH 5.0-5.1; mobile phase B=ACN. Mobile phases Aand B were filtered through 0.2μ filter paper. The column wasequilibrated with one column volume (about 20 mL) mobile phase A, andthe sample loaded. One column volume (about 20 mL) was run through thecolumn for desalting step, followed by the gradient below.

TABLE XX HPLC Mobile Phase Gradient Conditions Time (min.) % A % B 0.01100 0 40 40 60 45 0 100 60 Stop

The desired peak eluted between 20-32 minutes of the gradient run. Thepeak collected above was concentrated on a Rotavapor™ to ⅓ of itsoriginal volume and then freeze dried in a pre-weighed vial. About 7.0mg Conjugate 6 was obtained. This product was analyzed by HPLC (about70-80% pure by method B above) and by PAGE (Method C), where it ran as asingle band. See FIG. 11.

Example 6 Recognition of Naturally Processed HIV Epitopes

Splenic immune cells from mice immunized with Conjugate 3 (1 nmole) weresubjected to one round of in vitro stimulation and tested for theability to lyse HIV-infected CD4⁺ cells (R7 cells). R7 cells are capableof synthesizing HIV particles that express p24, a characteristicdiagnostic protein. The cells were tested at E:T ratios of 4, 20 and100. Results are shown in FIG. 12, which also shows results for controlJA2 cells. P (T<=t)=0.03 using a two-tail T test. The epitope recognizedafter immunization with Conjugate 3 is only a small portion of the totalamount of peptides presented on the surface of the cells. This assaydemonstrates recognition in one of the best in vitro systems todemonstrate that the immunization leads to recognition of bond fideHIV-infected cells. These results therefore indicate that the inventiveapproach to immunization is suitable for use in a clinical situation inwhich individuals are infected with HIV, for example for therapeuticuse, or for prophylaxis against HIV.

Example 7 Immunization Protects In Vivo Against Challenge with VacciniaVirus Expressing the HIV-Pol Gene

Three mice were immunized with 5 nmole Conjugate 3 or CpG Sequence #1826(control). Fourteen days after immunization, the mice were challenged byintraperitoneal administration of 1×10⁷ p.f.u. of recombinant vacciniavirus expressing the HIV-pol gene. After five days, the virus titer inthe ovaries of the mice was determined. Results are shown in FIG. 13.There was an approximate six orders of magnitude difference inprotection in the case of mice immunized with Conjugate 3 versuscontrol.

This study was repeated using intranasal immunization. See FIG. 14.These HLA A2/Kb mice were immunized with 1 nmole Conjugate 3, 10 nmolePADRE:I9V or 10 nmole CpG DNA. Fourteen days after immunization, allmice were challenged intranasally with 2×10⁷ p.f.u. recombinant vacciniavirus expressing HIV-pol. After five days, the virus titer in the miceovaries was determined. The animals that were administered 10 nmoles ofConjugate 3 were significantly better at providing protection againstthe vaccinia virus challenge than animals immunized with the PADRE:I9Vpeptide or animals receiving only CpG DNA without peptide (P=0.02;Wilcoxon two sample test). The results therefore indicate thatvaccination with Conjugate 3 protects against viral infection in vivo.

Example 8 Vaccination with Conjugate Versus Unconjugated Peptide

HLA A2/Kb mice were immunized both subcutaneously and intraperitoneallywith 1 nmole of Conjugate 3 or 1 nmole of PADRE:I9V peptide plus CpG DNA(unconjugated). Fourteen days later, the mice were challengedintraperitoneally with 1×10⁷ p.f.u. of recombinant vaccinia virusexpressing HIV-pol. After five days, the virus titer in the ovaries ofthe challenged mice was determined. Using a two-tailed T test, thedifference between these two vaccine strategies was significant atp<0.02. See FIG. 15. Immunization with one dose of 1 nmole of Conjugate3 provided protection against challenge with vaccinia virus expressingHIV-pol, whereas immunization with 1 nmole of the peptide withunconjugated CpG DNA did not confer protection against viral challenge.Ten-fold higher doses of unconjugated peptide vaccine were required toreach the same level of protection when the vaccine was administered asa conjugate.

A lower dose of Conjugate 3 (0.1 nmole) was tested in comparison to amixture of the peptide and CpG DNA. Two immunizations fourteen daysapart were administered to mice, followed by challenge with 1×10⁷ p.f.u.recombinant vaccinia virus expressing HIV-pol seven days after thesecond immunization. Five days later, the virus titer in the ovaries ofthe mice were determined. As is shown in FIG. 16, only the groupvaccinated with Conjugate 3 received significant protection (P<0.02;Wilcoxon two sample test). Animals receiving either peptide, CpG DNA orboth in combination were equivalently unprotected against viralchallenge. Conjugate 3 was ten times more sensitive than peptide plusDNA (unconjugated) for protection against vaccinia virus infection.

Conjugate 4, which is composed of CpG DNA #1826 (Oligo 3) linked using aHDA hydrazone linkage to PADRE followed by the HIV-gag CTL epitopeSLYNTVATL (SEQ ID NO:6), was tested as described for Conjugate 3 abovein comparison with unconjugated peptide. Chromium release assays andcytokine flow cytometry studies were carried out. One nanomole of theconjugate in a single immunization, followed by one in vitrostimulation, resulted in development of more than 23% HIV-gag CTLepitope-specific splenocytes (FIGS. 17B and 17A, respectively), greaterthan the mix of peptide plus DNA or DNA alone (FIG. 17C). This conjugatealso caused cytotoxicity against peptide-sensitized targets and againstthe R7 cell line.

Methods for flow cytometry were as follows. Mice were immunizedsubcutaneously with either 1 nmole Conjugate 4 or 1 nmole KSS:PADRE:S9L(SEQ ID NO:2) and 1 nmole CpG DNA. One day 14, splenocytes fromimmunized mice were stimulated in vitro for 7 days with irradiatedS9L-loaded LPS blasts from syngeneic litter mates. After the incubation,the cell mix was incubated further with S9L or irrelevant (control)peptide for six hours. The cells were passed through a Ficoll-Hypaque™gradient, and the live cells were washed with phosphate buffered saline.One million cells were stained with CD8-FITC, then fixed andpermeabilized by Perm/Fix™ solution for 30 minutes at 4° C. The cellsthen were washed in permeabilizing buffer and incubated with eitheranti-IFN-γ-APC or isotype-matched APC-labeled mAb. Two animals wereanalyzed for each vaccine and stimulation. See FIG. 17.

Example 9 HIV Immune Recognition after Multi-Epitope Peptide Vaccination

Conjugate 5 was synthesized to contain CpG DNA components in a linearchain with the CTL epitopes separated by a non-native asparagine residue(SEQ ID NOs:4 and 9). The conjugate was modified at the amino terminusas for Conjugates 3 and 4 and was physically evaluated by HPLC and gelelectrophoresis. The conjugate was dissolved in saline solution andadministered to HLA A2/Kb mice, and compared to a fusion peptide of allthree CTL epitopes mixed with CpG DNA or to CpG DNA alone. Eachvaccination contained 0.1 nmole vaccine component.

Fourteen days after vaccination, splenocytes from individual mice wereincubated with either I9V-loaded (FIG. 18) or S9L LPS blasts (FIG. 19under in vitro stimulation conditions. Cytolytic T cell responses weredetermined by chromium release assay using either I9V-loaded targets(FIG. 18) or S9L-loaded targets (FIG. 19). The data in FIG. 18 shows astatistically significant (P<0.03) difference between the conjugatedvaccine according to the invention and a mixture of the peptide and CpGDNA. Recognition of the S9L-loaded targets required a greater amount ofconjugate in the immunization (10 nmoles). Nevertheless, a statisticallysignificant (P<0.02) difference was found between the group immunizedwith Conjugate 5 versus the mixture of peptide plus DNA.

Additional targets were evaluated, including R7 cells that were infectedwith HIV. The level of recognition of the R7 target was higher with themulti-epitope peptide of Conjugate 5 than with Conjugate 3, even withoutconjugation. See FIG. 20. The comparison between the groups immunizedwith Conjugate 5 or a mixture of peptide plus DNA show that thedifference in recognition of the R7 cells is significantly greater withConjugate 5 (P<0.01). This demonstrates enhanced sensitivity ofrecognition of an HIV-infected cell line with Conjugate 5, and the levelof recognition is much higher than with any of the conjugates containingone CTL epitope examined above.

The conjugate strategy can be extended to a greater number of epitopesor different epitopes that cover or apply to a wide variety of HIVstrains and HLA types. Therefore, any single epitope or any combinationof epitopes can be included in the peptide portion of the conjugatevaccines according to this invention. For example, two or more CTLepitopes may be included as a linear chain, with or without spacersbetween the epitopes, or three, four, five, six or more epitopes asdesired to provide coverage for multi-ethnic populations, for differentviral strains, or both.

Example 10 Flow Cytometry Analysis of Immune Splenocytes

Aliquots of the splenocytes evaluated above were evaluated by flowcytometry for expression of IFN-γ and for quantitation of the frequencyof T cells specific for the relevant antigens. The immunizationdescribed in FIG. 18 was evaluated, with the modification that thesplenocytes, instead of being incubated with cellular targets, wereincubated with the I9V or irrelevant (control) peptide for six hours.The cells were passed through a Ficoll-Hypaque™ gradient and live cellswere washed with phosphate-buffered saline. One million of the cellswere stained with CD8-FITC. The cells then were fixed and permeabilizedusing Perm/Fix™ solution for 30 minutes at 4° C. Cells were washed inpermeabilizing buffer and incubated with eitheranti-IFN-γ-allophycocyanin (APC) or isotype-matched APC-labeled mAb. SeeFIGS. 21A, 21B, 21C and 21D.

The data show that there was limited non-specific reaction ofsplenocytes to either an isotype control (FIG. 21A) or an irrelevantpeptide (FIG. 21B), but that much more significant frequencies ofreactive T cells were found after exposure to the I9V peptide in thecase of immunization with the fusion peptide plus DNA, unconjugated(FIG. 20D), or with Conjugate 5 (FIG. 20C). The very high frequency ofreactive T cells in FIG. 20C demonstrates a good correlation with thechromium release assay of FIGS. 17-19. The activity of Conjugate 5 isvery high and affords a very high level of recognition by T cells, evenafter only one immunization. The extraordinary level of sensitivity inthis well-recognized in vivo model for human cellular and humoral immuneresponses demonstrates the merit of this immunization approach.

Example 11 Effect of T_(H) Epitope on Immunogenicity

Because the adjuvant activity of CpG was remarkable, experiments wereperformed to investigate the need for both CTL and T_(H) epitopecomponents in chimeric peptides for vaccine. Conjugate 7, which is CpG(oligo 3) covalently attached to the I9V peptide, was synthesized. SeeFIG. 3.

HHD II mice were immunized with 1 nmole of either Conjugate 7 orConjugate 3 (which is the same as Conjugate 7 but also contains PADRE).A chromium release assay was performed using I9V-loaded JA2 cells astargets. See FIG. 22 for results. Conjugate 7 had less activity thanConjugate 3 against these cells (p<0.005).

To address whether the defect in Conjugate 7 immunogenicity was themissing PADRE T_(H) epitope, immunizations with Conjugate 7 plus PADRET_(H) were compared to immunizations with Conjugate 7 alone. Each ofthree mice were immunized with Conjugate 7 alone, Conjugate 7 togetherwith the PADRE T_(H) epitope, Conjugate 3, or the PADRE:I9V fusionpeptide alone. FIG. 22 shows that the fusion peptide alone had littleimmunogenicity, while Conjugate 3 worked efficiently. Although Conjugate7 had minimal immunogenicity when used alone, in combination with thefree PADRE T_(H) epitope, its immunogenicity was restored almost to thelevel of Conjugate 3. See FIG. 22. This demonstrates that both the T_(H)and CTL epitopes should be present for maximal immunogenicity. Further,the majority of the stimulating activity of the PADRE epitope is causedby the sequence itself, rather than the artificial junction caused bychimerizing the CTL and T_(H) epitopes, since Conjugate 3 and Conjugate7 plus PADRE in trans have similar immunogenicity. Similar fusionpeptides, including a fusion peptide composed of PADRE and anHPV-specific CTL epitope, have been administered to volunteers and womenwith cervical neoplasia without causing observable autoimmunity

Example 12 Human-Specific ODN

Conjugate 10, composed of PADRE fused to I9V (SEQ ID NO:1), is identicalto Conjugate 3 except for the substitution of the CpG portion, which isa primate- and human-specific CpG-ODN (Oligo 4; SEQ ID NO:10). SeeHartmann et al., J. Immunol. 164:1617-1624, 2000. The chemical yield andpurity of this conjugate were similar to the others synthesized aboveusing CpG 1826 (murine). Groups of three HLA A2/Kb mice were immunizedwith either Conjugate 3 and Conjugate 10 (1 nmole). Chromium releaseassay results in FIG. 23 show that both conjugates were recognizedequivalently, providing additional evidence that these constructs arefeasible for use in a clinical setting in human patients. The CpG-ODNused in Conjugate 10 also is known under the trade names ProMune™ orVaxmune™ (Coley Pharmaceutical Group).

TABLE XXI Summary of Conjugate Structures and Sequences SEQ ID NAME NOSEQUENCE OR STRUCTURE PADRE:I9V 1AKXVAAWTLKAAAILKEPVHGV, X = cyclohexylalanine KSS:PADRE:59L 2KSSAKXVAAWTLKAAASLYNTVATL, X = cyclohexylalanine PADRE:I9V:K:S9L 3AKXVAAWTLKAAAILKEPVHGVKSLYNTVATL, X = cyclohexylalanine PADRE:I9V:N:S9L4 AKXVAAWTLKAAAILKEPVHGVNSLYNTVATL, X = cyclohexylalanine I9V 5ILKEPVHGV S9L 6 SLYNTVATL Oligo 1 7Phosphodiester single-stranded CpG DNA 5′-tccatgacgttcctgacgtt-3′ with the 5′ end modified to thiohexyl Oligo 2 8Phosphorothioated backbone, single-stranded CpGDNA 5′-tccatgacgttcctgacgtt-3′ with the 5′ end modified to thiohexylOligo 3 9 Phosphorothioated backbone, single-stranded CpG DNA 5′-H—CO—C₆H₃—CO—NH—(CH₂)₆-tccatgacgttcctgacgtt-3′(5′-aldehyde linker:Amidite-A ™) Oligo 4 105'-tcgtcgttttgtcgttttgtcgtt-3′ (phosphorothioate- substituted) Oligo 511 5'-ggGGGACGATCGTCgggggG-3′ (phosphorothioate-substituted at lower case letters) PADRE 12AKXVAAWTLKAAA, X = cyclohexylalanine Conjugate 1 NA PADRE-I9V + Oligo 1Conjugate 2 NA PADRE-I9V + Oligo 2 Conjugate 3 NA PADRE:I9V + Oligo 3Conjugate 4 NA KSS:PADRE:S9L + Oligo 3 Conjugate 5 NAPADRE:I9V:N:S9L + Oligo 3 Conjugate 6 NA PADRE:I9V:K:S9L + Oligo 3Conjugate 7 NA AAA:I9V + Oligo 3 Conjugate 10 NA PADRE:I9V + Oligo 4 HydNA NH₂—NH—C₆H₃N—CO—

REFERENCES

-   1. Cho et al., “IFN-alpha beta promote priming of antigen-specific    CD8+ and CD4+T lymphocytes by immunostimulatory DNA-based    vaccines.” J. Immunol. 168:4907-4913, 2002.-   2. Cho et al., “Immunostimulatory DNA-based vaccines induce    cytotoxic lymphocyte activity by a T-helper cell-independent    mechanism.” Nat. Biotechnol. 18:509-514, 2000.-   3. Hartmann et al., “Delineation of a CpG phosphorothioate    oligodeoxynucleotide for activation primate immune responses in    vitro and in vivo.” J. Immunol. 164:1617-1624, 2000.-   4. Homer et al., “Immunostimulatory DNA-based vaccines elicit    multifaceted immune responses against HIV at systemic and mucosal    sites.” J. Immunol. 167:1584-1591, 2001.-   5. Livingston et al., “The Hepatitis B virus-specific CTL responses    induced in humans by lipopeptide vaccination are comparable to those    elicited by acute viral infection.” J. Immunol. 159:1383-1392, 1997.-   6. Schirmbeck et al., “Antigenic epitopes fused to cationic peptide    bound to oligonucleotides facilitate toll-like receptor 9-dependent,    but CD4+ T cell help-independent priming of CD8+ T cells.” J.    Immunol. 171:5198-5207, 2003.-   7. Tighe et al., “Conjugation of protein to immunostimulatory DNA    results in rapid, long-lasting and potent induction of cell-mediated    and humoral immunity,” Eur. J. Immunol. 30(7):1939-1947, 2000.-   8. Tighe et al., “Conjugation of immunostimulatory DNA to the short    ragweed allergen and a 1 enhances its immunogenicity and reduces its    allergenicity.” J. Allergy Clin. Immunol. 106 (1 Pt. 1):124-134,    2000.-   9. Vitiello et al., “Development of a lipopeptide-based therapeutic    vaccine to treat chronic HBV infection. I. Induction of a primary    cytotoxic T lymphocyte response in humans.” J. Clin. Invest.    95:341-349, 1995.

The invention claimed is:
 1. A conjugated vaccine molecule whichcomprises a fusion peptide covalently attached to a DNA oligomer,wherein the fusion peptide comprises a T-help epitope and an antigenicpeptide CTL epitope, wherein the antigenic peptide CTL epitope isconjugated to the DNA oligomer, and wherein the antigenic peptide CTLepitope is amino acid residues 14-32 of SEQ ID NO:3 or amino acidresidues 14-32 of SEQ ID NO:4.
 2. The molecule of claim 1, wherein theDNA oligomer comprises a CpG sequence.
 3. The molecule of claim 2,wherein the DNA oligomer is a phosphodiester CpG DNA.
 4. The molecule ofclaim 2, wherein the DNA oligomer is a fully phosphorothioated backboneCpG DNA.
 5. The molecule of claim 2, wherein the DNA oligomer comprisesabout 8 to about 300 nucleotide bases.
 6. The molecule of claim 5,wherein the DNA oligomer comprises about 15 to about 100 nucleotidebases.
 7. The molecule of claim 6, wherein the DNA oligomer comprisesabout 20 to about 25 nucleotide bases.
 8. The molecule of claim 1,wherein fusion peptide is synthetic.